Diagnosis kit and chip for bladder cancer using bladder cancer specific methylation marker gene

ABSTRACT

The present invention relates to a kit and nucleic acid chip for diagnosing bladder cancer using a bladder cancer-specific marker gene. More particularly, the invention relates to a kit and nucleic acid chip for diagnosing bladder cancer, which can detect the promoter methylation of a bladder cancer-specific gene, the promoter or exon region of which is methylated specifically in transformed cells of bladder cancer. The use of the diagnostic kit or nucleic acid chip of the invention enables diagnosis of bladder cancer at an early stage of transformation, thus enabling early diagnosis of bladder cancer, and can diagnose bladder cancer in a more accurate and rapid manner compared to a conventional method.

TECHNICAL FIELD

The present invention relates to a kit and nucleic acid chip fordiagnosing bladder cancer using a bladder cancer-specific marker gene,and more particularly to a kit and nucleic acid chip for diagnosingbladder cancer, which can detect the promoter methylation of a bladdercancer-specific gene, the promoter region of which is methylatedspecifically in transformed cells of bladder cancer.

BACKGROUND ART

Bladder cancer is the most frequent cancer of the urinary system and wasfound to be caused by many factors. It is known that bladder cancer ismainly caused by smoking or various chemical substances (paints forleather, air pollutants, artificial sweetening agents, nitrates and thelike) which irritate the bladder wall while they are excreted as urineafter being absorbed in vivo.

As conventional methods for diagnosing bladder cancer, a method offinding abnormal cells in urine is used, but has low accuracy. Also,cystoscopy comprising inserting a catheter into the bladder andcollecting suspected tissue from the bladder is an invasive methodhaving relatively high accuracy.

Generally, when bladder cancer is diagnosed at an early stage, thesurvival rate of bladder cancer patients is increased, but it is noteasy to diagnose bladder cancer at an early stage. As a method fordiagnosing bladder cancer, a method of incising part of the body iscurrently being used, but it has difficulty in diagnosing bladder cancerat an early stage.

Bladder cancers are classified, according to invasion into the muscularlayer of the bladder, into superficial cancer and invasive cancer.Generally, about 30% of patients upon diagnosis of bladder cancer areinvasive bladder cancer patients. Thus, in order to increase thesurvival period of patients, it is the best method to diagnose bladdercancer at early stage when the bladder cancer lesions are small.Accordingly, there is an urgent need to development a diagnostic methodmore efficient than various prior diagnostic methods for bladder cancer,that is, a bladder cancer-specific biomarker which allows earlydiagnosis of bladder cancer, can treat a large amount of samples and hashigh sensitivity and specificity.

Recently, methods of diagnosing cancer through the measurement of DNAmethylation have been suggested. DNA methylation occurs mainly on thecytosine of CpG islands in the promoter region of a specific gene tointerfere with the binding of transcription factors, thus silencing theexpression of the gene. Thus, detecting the methylation of CpG islandsin the promoter of tumor inhibitory genes greatly assists in cancerresearch. Recently, an attempt has been actively made to determinepromoter methylation, by methods such as methylation-specific PCR(hereinafter referred to as MSP) or automatic DNA sequencing, for thediagnosis and screening of cancer.

Although there are disputes on whether the methylation of promoter CpGislands directly induces cancer development or causes a secondary changeafter cancer development, it has been found that tumor suppressor genes,DNA repair genes, cell cycle regulatory genes and the line in severalcancers are hyper-methylated, and thus the expression of these genes aresilenced. Particularly, it is known that the hyper-methylation of thepromoter region of a specific gene occurs at an early stage of cancerdevelopment.

Thus, the methylation of the promoter methylation of tumor-associatedgenes is an important indication of cancer and can be used in manyapplications, including the diagnosis and early diagnosis of cancer, theprediction of cancer development, the prediction of prognosis of cancer,follow-up examination after treatment, and the prediction of responsesto anticancer therapy. Recently, an actual attempt to examine thepromoter methylation of tumor-associated genes in blood, sputum, saliva,feces and to use the examined results for diagnosis and treatment ofvarious cancers has been actively made (Esteller, M. et al., CancerRes., 59:67, 1999; Sanchez-Cespedez, M. et al., Cancer Res., 60:892,2000; Ahlquist, D. A. et al., Gastroenterol., 119:1219, 2000).

Accordingly, the present inventors have made many efforts to develop adiagnostic kit capable of effectively diagnosing bladder cancer and, asa result, have found that bladder cancer can be diagnosed by measuringthe methylation degree using as a biomarker the promoter ofmethylation-associated genes which are expressed specifically in bladdercancer cells, thereby completing the present invention.

SUMMARY OF INVENTION

It is, therefore, an object of the present invention to provide a kitfor diagnosing bladder cancer, which comprises the methylated promoteror exon region of a bladder cancer marker gene.

Another object of the present invention is to provide a nucleic acidchip for diagnosing bladder cancer, which comprises a probe capable ofhybridizing with a fragment containing the CpG island of the bladdercancer-specific marker gene.

Still another object of the present invention is to provide a method formeasuring the methylation of the promoter or exon region of a geneoriginated from a clinical sample.

To achieve the above objects, the present invention provides a kit fordiagnosing bladder cancer, which comprises the methylated promoter orexon region of a bladder cancer marker gene selected from the groupconsisting of: (1) CDX2 (NM_(—)001265)—caudal type homeoboxtranscription factor 2; (2) CYP1B1 (NM_(—)000104)—cytochrome P450,family 1, subfamily B, polypeptide 1; (3) VSX1 (NM_(—)199425)—visualsystem homeobox 1 homolog, CHX10-like (zebrafish); (4) HOXA11(NM_(—)005523)—homeobox A11; (5) T (NM_(—)003181)—T, brachyury homolog(mouse); (6) TBX5 (NM_(—)080717)—T-box 5; (7) PENK(NM_(—)006211)—proenkephalin; (8) PAQR9 (NM_(—)198504)—progestin andadipoQ receptor family member IV; (9) LHX2 (NM_(—)004789)—LIM Homeobox2; and (10) SIM2 (U80456)—single-minded homog 2 (Drosophila).

The present invention also provides a nucleic acid chip for diagnosingbladder cancer, which comprises a probe capable of hybridizing with afragment containing the CpG island of the promoter or exon region of thebladder cancer marker gene selected from the group consisting of: (1)CDX2 (NM_(—)001265)—caudal type homeobox transcription factor 2; (2)CYP1B1 (NM_(—)000104)—cytochrome P450, family 1, subfamily B,polypeptide 1; (3) VSX1 (NM_(—)199425)—visual system homeobox 1 homolog,CHX10-like (zebrafish); (4) HOXA11 (NM_(—)005523)—homeobox A11; (5) T(NM_(—)003181)—T, brachyury homolog (mouse); (6) TBX5(NM_(—)080717)—T-box 5; (7) PENK (NM_(—)006211)—proenkephalin; (8) PAQR9(NM_(—)198504)—progestin and adipoQ receptor family member IV; (9) LHX2(NM_(—)004789)—LIM Homeobox 2; and (10) SIM2 (U80456)—single-mindedhomog 2 (Drosophila).

The present invention also provides a method for detecting themethylation of the promoter or exon region of a clinicalsample-originated gene selected from the group consisting of CDX2,CYP1B1, VSX1, HOXA11, T, TBX5, PENK, PAQR9, LHX2 and SIM2.

Other features and embodiments of the present invention will be moreapparent from the following detailed description and the appendedclaims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram showing a process of discovering amethylated biomarker for diagnosis of bladder cancer from the urinarycells of normal persons and bladder cancer patients through CpGmicrroarray analysis.

FIG. 2 quantitatively shows the methylation degree obtained throughpyrosequencing of 10 methylation biomarkers in bladder cancer celllines.

FIG. 3(A) shows measurement results for the methylation indexes of theCDX2, the CYP1B1 and the T biomarker genes in clinical samples. FIG.3(A) shows measurement results for the methylation degrees of the CDX2,the CYP1B1 and the T biomarker genes in the urinary cells of normalpersons, Cystitis patients, hematuria patients and bladder cancerpatients.

FIG. 3(B) shows measurement results for the methylation indexes of theTBX5, the LHX2 and the SIM2 biomarker genes in clinical samples. FIG.3(B) shows measurement results for the methylation degrees of the TBX5,the LHX2 and the SIM2 biomarker genes in the urinary cells of normalpersons, Cystitis patients, hematuria patients and bladder cancerpatients.

FIG. 3(C) shows measurement results for the methylation indexes of theVSX1, the HOXA11 and the PENK biomarker genes in clinical samples. FIG.3(C) shows measurement results for the methylation degrees of the VSX1,the HOXA11 and the PENK biomarker genes in the urinary cells of normalpersons, Cystitis patients, hematuria patients and bladder cancerpatients.

FIG. 3(D) shows measurement results for the methylation indexes of thePAQR9 biomarker genes in clinical samples. FIG. 3(D) shows measurementresults for the methylation degrees of the PAQR9 biomarker genes in theurinary cells of normal persons, Cystitis patients, hematuria patientsand bladder cancer patients.

FIG. 4 a shows the results of receiver operating characteristic (ROC)curve analysis conducted to measure the sensitivity and specificity ofthe CDX2 and the CYP1B1 methylation biomarkers for diagnosis of bladdercancer.

FIG. 4 b shows the results of receiver operating characteristic (ROC)curve analysis conducted to measure the sensitivity and specificity ofthe VSX1 and the HOXA11 methylation biomarkers for diagnosis of bladdercancer.

FIG. 4 c shows the results of receiver operating characteristic (ROC)curve analysis conducted to measure the sensitivity and specificity ofthe T and the TBX5 methylation biomarkers for diagnosis of bladdercancer.

FIG. 4 d shows the results of receiver operating characteristic (ROC)curve analysis conducted to measure the sensitivity and specificity ofthe PENK and the PAQR9 methylation biomarkers for diagnosis of bladdercancer.

FIG. 4 e shows the results of receiver operating characteristic (ROC)curve analysis conducted to measure the sensitivity and specificity ofthe LHX2 and the SIM2 methylation biomarkers for diagnosis of bladdercancer.

FIG. 5 shows the frequency of methylation in the urinary cells of normalpersons and bladder cancer patients.

FIGS. 6(A)-6(D) shows the methylation profile of an optimal panel of 6biomarker genes for bladder cancer diagnosis (FIG. 6(A)), selected fromamong 10 biomarkers using logistic regression analysis, and shows thesensitivity and specificity of the gene panel for diagnosis of bladdercancer (FIG. 6(B)).

FIG. 7 shows the results of PCR performed using the methylatedDNA-specific binding protein MBD in order to measure the methylation ofthe biomarker SIM2 gene for bladder cancer cell in bladder cancer celllines.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

In one aspect, the present invention relates to a kit for diagnosingbladder cancer, which comprises the methylated promoter or exon regionof a bladder cancer marker gene.

In another aspect, the present invention relates to a nucleic acid chipfor diagnosing bladder cancer, which comprises a probe capable ofhybridizing with a fragment containing the CpG island of the promoter orexon region of a bladder cancer marker gene.

In the present invention, the promoter or exon region may contain atleast one methylated CpG dinucleotide. Also, the promoter or exon regionis any one of DNA sequences represented in SEQ ID NO: 31 to SEQ ID NO:40.

In the present invention, the probe preferably has a size ranging from10 bp to 1 kb, and has a homology with a base sequence containing theCpG island of the promoter or exon region of a bladder cancer markergene, such that it can hybridize with the base sequence. Morepreferably, the probe has a size of 10-100 bp, and has a homology with abase sequence containing the CpG island of the promoter or exon regionof a bladder cancer marker gene, such that it can hybridize with thebase sequence in strict conditions. If the size of the probe is lessthan 10 bp, non-specific hybridization will occur, and if it is morethan 1 kb, the binding between the probes will occur, thus making itdifficult to read hybridization results.

A method for screening a methylation marker gene according to thepresent invention comprises the steps of: (a) isolating genomic DNAsfrom transformed cells and non-transformed cells; (b) reacting theisolated genomic DNAs to with a protein binding to methylated DNA andisolating methylated DNAs from the genomic DNAs; and (c) amplifying theisolated methylated DNAs, hybridizing the amplified DNAs to CpGmicroarrays, and selecting a methylation marker gene showing thegreatest difference in methylation degree between normal cells andcancer cells among from the hybridized genes.

By the method for screening the methylation biomarker gene, it ispossible to screen various genes, which are methylated not only inbladder cancer, but also in various dysplasic stages which progress tobladder cancer. The screened genes are also useful for blood cancerscreening, risk assessment, prognosis, disease identification, diseasestaging, and selection of therapeutic targets.

The identification of the methylated gene in bladder cancer andabnormalities at various stages enables early diagnosis of bladdercancer in an accurate and effective manner, and allows establishment ofmethylation data using multiple genes and identification of newtherapeutic targets. Additionally, methylation data according to thepresent invention enables establishment of a more accurate system fordiagnosing bladder cancer, when it is used together with a method fordetecting other non-methylation-associated biomarkers.

The inventive method enables diagnosis of bladder cancer progression atvarious stages by determining the methylation stage of at least onenucleic acid biomarker obtained from a sample. When the methylationstage of nucleic acid isolated from a sample at each stage of bladdercancer is compared with the methylation stage of at least one nucleicacid obtained from a sample having no abnormality in the cellproliferation of bladder tissue, a certain stage of bladder cancer inthe sample can be determined. The methylation stage may behypermethylation.

In one embodiment of the present invention, nucleic acid can bemethylated in the regulatory region of a gene. In another embodiment,since methylation begins from the outer boundary of the regulatoryregion of a gene and then spreads inward, detection of methylation atthe outer boundary of the regulatory region enables early diagnosis ofgenes which are involved in cell transformation.

In still another embodiment of the present invention, the cell growthabnormality (dysplasia) of bladder tissue can be diagnosed by detectingthe methylation of at least one nucleic acid of the following nucleicacids using a kit or a nucleic acid chip: CDX2 (NM_(—)001265, caudaltype homeobox transcription factor 2); CYP1B1 (NM_(—)000104, cytochromeP450, family 1, subfamily B, polypeptide 1); VSX1 (NM_(—)199425, visualsystem homeobox 1 homolog, CHX10-like (zebrafish)); HOXA11(NM_(—)005523, homeobox A11); T (NM_(—)003181, T, brachyury homolog(mouse)); TBX5 (NM_(—)080717, T-box 5); PENK (NM_(—)006211,proenkephalin); and PAQR9 (NM_(—)198504, progestin and adipoQ receptorfamily member IV); LHX2 (NM_(—)004789) LIM Homeobox 2; SIM2 (U80456),single-minded homog 2 (Drosophila) gene and combination thereof.

The use of the diagnostic kit or nucleic acid chip of the presentinvention can determine the cell growth abnormality of bladder tissue ina sample. The method for determining the cell growth abnormality ofbladder tissue comprises determining the methylation of at least onenucleic acid isolated from a sample. In the method, the methylationstage of at least one nucleic acid is compared with the methylationstage of a nucleic acid isolated from a sample having no cell growthabnormality (dysplasia).

The examples of said nucleic acid are follows: CDX2 (NM_(—)001265,caudal type homeobox transcription factor 2); CYP1B1 (NM_(—)000104,cytochrome P450, family 1, subfamily B, polypeptide 1); VSX1(NM_(—)199425, visual system homeobox 1 homolog, CHX10-like(zebrafish)); HOXA11 (NM_(—)005523, homeobox A11); T (NM_(—)003181, T,brachyury homolog (mouse)); TBX5 (NM_(—)080717, T-box 5); PENK(NM_(—)006211, proenkephalin); and PAQR9 (NM_(—)198504, progestin andadipoQ receptor family member IV); LHX2 (NM_(—)004789) LIM Homeobox 2;SIM2 (U80456), single-minded homog 2 (Drosophila) gene and combinationthereof.

In still another embodiment of the present invention, cells capable offorming bladder cancer can be diagnosed at an early stage using themethylation gene marker. When genes confirmed to be methylated in cancercells are methylated in cells which seem to be normal clinically ormorphologically, the cells that seem to be normal are cells, thecarcinogenesis of which is in progress. Thus, bladder cancer can bediagnosed at an early stage by detecting the methylation of bladdercancer-specific genes in the cells that seem to be normal.

The use of the methylation marker gene of the present invention enablesdetection of the cell growth abnormality (dysplasia progression) ofbladder tissue in a sample. The method for detecting the cell growthabnormality (dysplasia progression) of bladder tissue comprises bringingat least one nucleic acid isolated from a sample into contact with anagent capable of determining the methylation status of the nucleic acid.The method comprises determining the methylation status of at least oneregion in at least one nucleic acid, and the methylation status of thenucleic acid differs from the methylation status of the same region in anucleic acid isolated from a sample having no cell growth abnormality(dysplasia progression) of bladder tissue.

In still another embodiment of the present invention, transformedbladder cancer cells can be detected by examining the methylation of amarker gene using the above-described kit or nucleic acid chip.

In still another embodiment of the present invention, bladder cancer canbe diagnosed by examining the methylation of a marker gene using theabove-described kit or nucleic acid chip.

In still another embodiment of the present invention, the likelihood ofprogression to bladder cancer can be diagnosed by examining themethylation of a marker gene with the above-described kit or nucleicacid chip in a sample showing a normal phenotype. The sample may besolid or liquid tissue, cell, urine, serum or plasma.

In still another aspect, the present invention relates to a method fordetecting the promoter methylation of a clinical sample-originated gene.

In the present invention, the method for measuring the promotermethylation of a clinical sample-originated gene may be selected fromthe group consisting of PCR, methylation specific PCR, real-timemethylation specific PCR, PCR using a methylated DNA-specific bindingprotein, quantitative PCR, pyrosequencing and bisulfite sequencing, andthe clinical sample is preferably a tissue, cell, blood or urineoriginated from patients suspected of cancer or subjects to bediagnosed.

In the present invention, the method for detecting the promotermethylation of the gene comprises the steps of: (a) isolating a sampleDNA from a clinical sample; (b) amplifying the isolated DNA with primerscapable of amplifying a fragment containing the promoter CpG island of agene selected from the group consisting of CDX2, CYP1B1, VSX1, HOXA11,T, TBX5, PENK, PAQR9, LHX2 and SIM2; and (c) determining the promotermethylation of the DNA on the basis of whether the DNA has beenamplified or not in step (b).

In another embodiment of the present invention, the likelihood ofdevelopment of tissue to bladder cancer can be evaluated by examiningthe methylation frequency of a gene which is methylated specifically inbladder cancer and determining the methylation frequency of tissuehaving the likelihood of progression to bladder cancer.

As used herein, “cell conversion” refers to the change incharacteristics of a cell from one form to another such as from normalto abnormal, non-tumorous to tumorous, undifferentiated todifferentiated, stem cell to non-stem cell. Further, the conversion maybe recognized by morphology of the cell, phenotype of the cell,biochemical characteristics and so on.

As used herein, the term “early diagnosis” of cancer refers todiscovering the likelihood of cancer before metastasis. Preferably, itrefers to discovering the likelihood of cancer before a morphologicalchange in a sample tissue or cell is observed. Additionally, the term“early diagnosis” of transformation the high probability of a cell toundergo transformation in its early stages before the cell ismorphologically designated as being transformed.

As used herein, the term “hypermethylation” refers to the methylation ofCpG islands.

As used herein, the term “sample” or “biological sample” is referred toin its broadest sense, and includes any biological sample obtained froman individual, body fluid, cell line, tissue culture or other sources,according to the type of analysis that is to be performed. Methods ofobtaining body fluid and tissue biopsy from mammals are generally widelyknown. A preferred source is bladder biopsy.

Screening for Methylation Regulated Biomarkers

The present invention is directed to a method of determining biomarkergenes that are methylated when the cell or tissue is converted orchanged from one type of cell to another. As used herein, “converted”cell refers to the change in characteristics of a cell or tissue fromone form to another such as from normal to abnormal, non-tumorous totumorous, undifferentiated to differentiated and so on.

In one Example of the present invention, urinary cells were isolatedfrom the urine of normal persons and bladder cancer patients, and thengenomic DNAs were isolated from the urinary cells. In order to obtainonly methylated DNAs from the genomic DNAs, the genomic DNAs wereallowed to react with McrBt binding to methylated DNA, and thenmethylated DNAs binding to the McrBt protein were isolated. The isolatedmethylated DNAs binding to the McrBt protein were amplified, and thenthe DNAs originated from the normal persons were labeled with Cy3, andthe DNAs originated from the bladder cancer patients were labeled withCy5. Then, the DNAs were hybridized to human CpG-island microarrays, andgenes showing the greatest difference in methylation degree between thenormal persons and the bladder cancer patients were selected asbiomarkers.

In the present invention, in order to further confirm whether the 10biomarkers have been methylated, pyrosequencing was performed.

Specifically, total genomic DNA was isolated from the bladder cell linesRT-4, J82, HT1197 and HT1376 and treated with bisulfite. The genomic DNAconverted with bisulfite was amplified. Then, the amplified PCR productwas subjected to pyrosequencing in order to measure the methylationdegree of the genes. As a result, it could be seen that the 10biomarkers were all methylated.

Biomarker for Bladder Cancer

The present invention provides a biomarker for diagnosing bladdercancer.

Biomarkers for Bladder Cancer—Using Cancer Cells for Comparison withNormal Cells

In one embodiment of the present invention, it is understood that“normal” cells are those that do not show any abnormal morphological orcytological changes. “Tumor” cells mean cancer cells. “Non-tumor” cellsare those cells that were part of the diseased tissue but were notconsidered to be the tumor portion.

In one aspect, the present invention is based on the relationshipbetween bladder cancer and the hypermethylation of the promoter or exonregion of the following 10 genes: CDX2 (NM_(—)001265, caudal typehomeobox transcription factor 2); CYP1B1 (NM_(—)000104, cytochrome P450,family 1, subfamily B, polypeptide 1); VSX1 (NM_(—)199425, visual systemhomeobox 1 homolog, CHX10-like (zebrafish)); HOXA11 (NM_(—)005523,homeobox A11); T (NM_(—)003181, T, brachyury homolog (mouse)); TBX5(NM_(—)080717, T-box 5); PENK (NM_(—)006211, proenkephalin); and PAQR9(NM_(—)198504, progestin and adipoQ receptor family member IV); LHX2(NM_(—)004789)—LIM Homeobox 2; and SIM2 (U80456)—single-minded homolog 2(Drosophila); gene.

With other applications of the diagnostic kit or nucleic acid chip ofthe present invention, the invention can diagnose a cellularproliferative disorder of bladder tissue in a subject by determining thestate of methylation of one or more nucleic acids isolated from thesubject, wherein the state of methylation of one or more nucleic acidsas compared with the state of methylation of one or more nucleic acidsfrom a subject not having the cellular proliferative disorder of bladdertissue is indicative of a cellular proliferative disorder of bladdertissue in the subject. A preferred nucleic acid is a CpG-containingnucleic acid, such as a CpG island.

With other applications of the diagnostic kit or nucleic acid chip ofthe present invention, the cell growth abnormality of bladder tissue ina subject can be diagnosed comprising determining the methylation of oneor more nucleic acids isolated from the subject. Said nucleic acid ispreferably encoding the followings: CDX2 (NM_(—)001265, caudal typehomeobox transcription factor 2); CYP1B1 (NM_(—)000104, cytochrome P450,family 1, subfamily B, polypeptide 1); VSX1 (NM_(—)199425, visual systemhomeobox 1 homolog, CHX10-like (zebrafish)); HOXA11 (NM_(—)005523,homeobox A11); T (NM_(—)003181, T, brachyury homolog (mouse)); TBX5(NM_(—)080717, T-box 5); PENK (NM_(—)006211, proenkephalin); and PAQR9(NM_(—)198504, progestin and adipoQ receptor family member IV); LHX2(NM_(—)004789)—LIM Homeobox 2; and SIM2 (U80456)—single-minded homolog 2(Drosophila); gene and combinations thereof. The state of methylation ofone or more nucleic acids as compared with the state of methylation ofsaid nucleic acid from a subject not having a predisposition to thecellular proliferative disorder of bladder tissue is indicative of acell proliferative disorder of bladder tissue in the subject.

As used herein, “predisposition” refers to an increased likelihood thatan individual will have a disorder. Although a subject with apredisposition does not yet have the disorder, there exists an increasedpropensity to the disease.

Another embodiment of the invention provides a method for diagnosing acellular proliferative disorder of bladder tissue in a subjectcomprising contacting a nucleic acid-containing specimen from thesubject with an agent that provides a determination of the methylationstate of nucleic acids in the specimen, and identifying the methylationstate of at least one region of at least one nucleic acid, wherein themethylation state of at least one region of at least one nucleic acidthat is different from the methylation state of the same region of thesame nucleic acid in a subject not having the cellular proliferativedisorder is indicative of a cellular proliferative disorder of bladdertissue in the subject.

The inventive method includes determining the state of methylation ofone or more regions of one or more nucleic acids isolated from thesubject. The phrases “nucleic acid” or “nucleic acid sequence” as usedherein refer to an oligonucleotide, nucleotide, polynucleotide, or to afragment of any of these, to DNA or RNA of genomic or synthetic originwhich may be single-stranded or double-stranded, to DNA or RNA ofgenomic or synthetic origin which may represent a sense or antisensestrand, peptide nucleic acid (PNA), or to any DNA-like or RNA-likematerial of natural or synthetic origin. As will be understood by thoseof skill in the art, when the nucleic acid is RNA, the deoxynucleotidesA, G, C, and T are replaced by ribonucleotides A, G, C, and U,respectively.

The nucleic acid of interest can be any nucleic acid where it isdesirable to detect the presence of a differentially methylated CpGisland. The CpG island is a CpG rich region of a nucleic acid sequence.

Methylation

Any nucleic acid sample, in purified or nonpurified form, can beutilized in accordance with the present invention, provided it containsor is suspected of containing, a nucleic acid sequence containing atarget locus (e.g., CpG-containing nucleic acid). One nucleic acidregion capable of being differentially methylated is a CpG island, asequence of nucleic acid with an increased density relative to othernucleic acid regions of the dinucleotide CpG. The CpG doublet occurs invertebrate DNA at only about 20% of the frequency that would be expectedfrom the proportion of G*C base pairs. In certain regions, the densityof CpG doublets reaches the predicted value; it is increased by ten foldrelative to the rest of the genome. CpG islands have an average G*Ccontent of about 60%, and general DNA have an average G*C contents ofabout 40%. The islands take the form of stretches of DNA typically aboutone to two kilobases long. There are about 45,000 such islands in thehuman genome.

In many genes, the CpG islands begin just upstream of a promoter andextend downstream into the transcribed region. Methylation of a CpGisland at a promoter usually prevents expression of the gene. Theislands can also surround the 5′ region of the coding region of the geneas well as the 3′ region of the coding region. Thus, CpG islands can befound in multiple regions of a nucleic acid sequence including upstreamof coding sequences in a regulatory region including a promoter region,in the coding regions (e.g., exons), in downstream of coding regions,for example, enhancer regions, and in introns.

In general, the CpG-containing nucleic acid is DNA. However, inventionmethods may employ, for example, samples that contain DNA, or DNA andRNA, including messenger RNA, wherein DNA or RNA may be single strandedor double stranded, or a DNA-RNA hybrid may be included in the sample.

A mixture of nucleic acids may also be employed. The specific nucleicacid sequence to be detected may be a fraction of a larger molecule orcan be present initially as a discrete molecule, so that the specificsequence constitutes the entire nucleic acid. It is not necessary thatthe nucleic acid sequence is present initially in a pure form, thenucleic acid may be a minor fraction of a complex mixture, such ascontained in whole human DNA. The nucleic acid-containing sample usedfor determination of the state of methylation of nucleic acids containedin the sample or detection of methylated CpG islands may be extracted bya variety of techniques such as that described by Sambrook, et al.(Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989;incorporated in its entirety herein by reference).

A nucleic acid can contain a regulatory region which is a region of DNAthat encodes information or controls transcription of the nucleic acid.Regulatory regions include at least one promoter. A “promoter” is aminimal sequence sufficient to direct transcription, to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents. Promotersmay be located in the 5′ or 3′ regions of the gene. Promoter regions, inwhole or in part, of a number of nucleic acids can be examined for sitesof CpG-island methylation. Moreover, it is generally recognized thatmethylation of the target gene promoter proceeds naturally from theouter boundary inward. Therefore, early stage of cell conversion can bedetected by assaying for methylation in these outer areas of thepromoter region.

Nucleic acids isolated from a subject are obtained in a biologicalspecimen from the subject. If it is desired to detect bladder cancer orstages of bladder cancer progression, the nucleic acid may be isolatedfrom bladder tissue by scraping or taking a biopsy. These specimens maybe obtained by various medical procedures known to those of skill in theart.

In one aspect of the invention, the state of methylation in nucleicacids of the sample obtained from a subject is hypermethylation comparedwith the same regions of the nucleic acid in a subject not having thecellular proliferative disorder of bladder tissue. Hypermethylation, asused herein, is the presence of methylated alleles in one or morenucleic acids. Nucleic acids from a subject not having a cellularproliferative disorder of bladder tissues contain no detectablemethylated alleles when the same nucleic acids are examined.

Sample

The present invention describes early diagnosis of bladder cancer andutilizes the methylation of bladder cancer-specific genes. Themethylation of bladder cancer-specific genes also occurred in tissuenear tumor sites. Therefore, in the method for early diagnosis ofbladder cancer, the methylation of bladder cancer-specific genes can bedetected by examining all samples including liquid or solid tissue. Thesamples include, but are not limited to, tissue, cell, urine, serum orplasma.

Individual Genes and Panel

It is understood that the present invention may be practiced using eachgene separately as a diagnostic or prognostic marker, or a few markergenes combined into a panel display format so that several marker genesmay be detected to increase reliability and efficiency. Further, any ofthe genes identified in the present application may be used individuallyor as a set of genes in any combination with any of the other genes thatare recited in the application. Also, genes may be ranked and weightedaccording to their importance together with the number of genes that aremethylated, and a level of likelihood of development to cancer can beassigned. Such algorithms are within the scope of the present invention.

Methylation Detection Methods Methylation Specific PCR

When genomic DNA is treated with bisulfite, the methylated cytosine inthe 5′-CpG'-3 region remains without changes, and unmethylated cytosineis changed to uracil. Thus, for a base sequence modified by bisulfitetreatment, PCR primers corresponding to regions in which a 5′-CpG-3′base sequence is present were constructed. Herein, two kinds of primerscorresponding to the methylated case and the unmethylated case wereconstructed. When genomic DNA is modified with bisulfite and thensubjected to PCR using the two kinds of primers, in the case in whichthe DNA is methylated, a PCR product is made from the DNA in which theprimers corresponding to the methylated base sequence are used. Incontrast, in the case in which the gene is unmethylated, a PCR productis made from the DNA in which the primers corresponding to theunmethylated base sequence are used. The methylation of DNA can bequalitatively analyzed using agarose gel electrophoresis.

Real-Time Methylation-Specific PCR

Real-time methylation-specific PCR is a real-time measurement methodmodified from methylation-specific PCR, and comprises treating genomicDNA with bisulfite, designing PCR primers corresponding to themethylated case and performing real-time PCR using the primers. Herein,methods of detecting methylation include two methods: a method ofperforming detection using a TanMan probe complementary to the amplifiedbase sequence, and a method of performing detection using Sybergreen.Thus, real-time methylation-specific PCR selectively quantitativelyanalyze only DNA. Herein, a standard curve was prepared using an invitro methylated DNA sample, and for standardization, a gene having no5′-CpG-3′ sequence in the base sequence was also amplified as a negativecontrol group and was quantitatively analyzed for the methylationdegree.

Pyrosequencing

Pyrosequencing is a real-time sequencing method modified from abisulfite sequencing method. In the same manner as bisulfite sequencing,genomic DNA was modified by bisulfite treatment, and then primerscorresponding to a region having no 5′-CpG-3′ base sequence wereconstructed. After the genomic DNA had been treated with bisulfite, itwas amplified with the PCR primers, and then subjected to real-timesequence analysis using sequencing primers. The amounts of cytosine andthymine in the 5′-CpG-3′ region were quantitatively analyzed, and themethylation degree was expressed as a methylation index.

PCR or Quantitative PCR Using Methylated DNA-Specific Binding Proteinand DNA Chip

In a PCR or DNA chip method using a methylated DNA-specific bindingprotein, when a protein binding specifically only to methylated DNA ismixed with DNA, the protein binds specifically only to methylated DNA,and thus only methylated DNA can be isolated. In the present invention,genomic DNA was mixed with a methylated DNA-specific binding protein,and then only methylated DNA was selectively isolated. The isolated DNAwas amplified using PCR primers corresponding to the promoter regionthereof, and then the methylation of the DNA was measured by agarose gelelectrophoresis.

In addition, the methylation of DNA can also be measured by aquantitative PCR method. Specifically, methylated DNA isolated using amethylated DNA-specific binding protein can be labeled with afluorescent dye and hybridized to a DNA chip in which complementaryprobes are integrated, thus measuring the methylation of the DNA.Herein, the methylated DNA-specific binding protein is not limited toMcrBt.

Detection of Differential Methylation-Methylation Sensitive RestrictionEndonuclease

Detection of differential methylation can be accomplished by contactinga nucleic acid sample with a methylation sensitive restrictionendonuclease that cleaves only unmethylated CpG sites under conditionsand for a time to allow cleavage of unmethylated nucleic acid.

In a separate reaction, the sample is further contacted with anisoschizomer of the methylation sensitive restriction endonuclease thatcleaves both methylated and unmethylated CpG-sites under conditions andfor a time to allow cleavage of methylated nucleic acid.

Specific primers are added to the nucleic acid sample under conditionsand for a time to allow nucleic acid amplification to occur byconventional methods. The presence of amplified product in the sampledigested with methylation sensitive restriction endonuclease but absenceof an amplified product in sample digested with an isoschizomer of themethylation sensitive restriction enzyme endonuclease that cleaves bothmethylated and unmethylated CpG-sites indicates that methylation hasoccurred at the nucleic acid region being assayed. However, lack ofamplified product in the sample digested with methylation sensitiverestriction endonuclease together with lack of an amplified product inthe sample digested with an isoschizomer of the methylation sensitiverestriction enzyme endonuclease that cleaves both methylated andunmethylated CpG-sites indicates that methylation has not occurred atthe nucleic acid region being assayed.

As used herein, a “methylation sensitive restriction endonuclease” is arestriction endonuclease that includes CG as part of its recognitionsite and has altered activity when the C is methylated as compared towhen the C is not methylated (e.g., Sma I). Non-limiting examples ofmethylation sensitive restriction endonucleases include MspI, HpaII,BssHII, BstUI and NotI. Such enzymes can be used alone or incombination. Other methylation sensitive restriction endonucleases suchas SacII and EagI may be applied to the present invention, but are notlimited to these enzymes.

An “isoschizomer” of a methylation sensitive restriction endonuclease isa restriction endonuclease that recognizes the same recognition site asa methylation sensitive restriction endonuclease but cleaves bothmethylated CGs and unmethylated CGs, such as for example, MspI.

Primers of the invention are designed to be “substantially”complementary to each strand of the locus to be amplified and includethe appropriate G or C nucleotides as discussed above. This means thatthe primers must be sufficiently complementary to hybridize with theirrespective strands under conditions that allow the agent forpolymerization to perform. Primers of the invention are employed in theamplification process, which is an enzymatic chain reaction thatproduces exponentially increasing quantities of target locus relative tothe number of reaction steps involved (e.g., polymerase chain reaction(PCR)). Typically, one primer is complementary to the negative (−)strand of the locus (antisense primer) and the other is complementary tothe positive (+) strand (sense primer). Annealing the primers todenatured nucleic acid followed by extension with an enzyme, such as thelarge fragment of DNA Polymerase I (Klenow) and nucleotides, results innewly synthesized + and − strands containing the target locus sequence.Because these newly synthesized sequences are also templates, repeatedcycles of denaturing, primer annealing, and extension results inexponential production of the region (i.e., the target locus sequence)defined by the primer. The product of the chain reaction is a discretenucleic acid duplex with termini corresponding to the ends of thespecific primers employed.

Preferably, the method of amplifying is by PCR, as described herein andas is commonly used by those of ordinary skill in the art. However,alternative methods of amplification have been described and can also beemployed such as real time PCR or linear amplification using isothermalenzyme. Multiplex amplification reactions may also be used.

Detection of Differential Methylation-Bifulfite Sequencing Method

Another method for detecting a methylated CpG-containing nucleic acidincludes contacting a nucleic acid-containing specimen with an agentthat modifies unmethylated cytosine, amplifying the CpG-containingnucleic acid in the specimen by means of CpG-specific oligonucleotideprimers, wherein the oligonucleotide primers distinguish betweenmodified methylated and non-methylated nucleic acid and detecting themethylated nucleic acid. The amplification step is optional and althoughdesirable, is not essential. The method relies on the PCR reactionitself to distinguish between modified (e.g., chemically modified)methylated and unmethylated DNA. Such methods are described in U.S. Pat.No. 5,786,146, the contents of which are incorporated herein in theirentirety especially as they relate to the bisulfate sequencing methodfor detection of methylated nucleic acid.

Substrates

Once the target nucleic acid region is amplified, the nucleic acid canbe hybridized to a known gene probe immobilized on a solid support todetect the presence of the nucleic acid sequence.

As used herein, “substrate,” when used in reference to a substance,structure, surface or material, means a composition comprising anonbiological, synthetic, nonliving, planar, spherical or flat surfacethat is not heretofore known to comprise a specific binding,hybridization or catalytic recognition site or a plurality of differentrecognition sites or a number of different recognition sites whichexceeds the number of different molecular species comprising thesurface, structure or material. The substrate may include, for exampleand without limitation, semiconductors, synthetic (organic) metals,synthetic semiconductors, insulators and dopants; metals, alloys,elements, compounds and minerals; synthetic, cleaved, etched,lithographed, printed, machined and microfabricated slides, devices,structures and surfaces; industrial polymers, plastics, membranes;silicon, silicates, glass, metals and ceramics; wood, paper, cardboard,cotton, wool, cloth, woven and nonwoven fibers, materials and fabrics.

Several types of membranes are known to one of skill in the art foradhesion of nucleic acid sequences. Specific non-limiting examples ofthese membranes include nitrocellulose or other membranes used fordetection of gene expression such as polyvinylchloride, diazotized paperand other commercially available membranes such as GENESCREEN™,ZETAPROBE™ (Biorad), and NYTRAN™. Beads, glass, wafer and metalsubstrates are included. Methods for attaching nucleic acids to theseobjects are well known to one of skill in the art. Alternatively,screening can be done in liquid phase.

Hybridization Conditions

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofhomology, nucleotide sequence composition (e.g., GC/AT content), andnucleic acid type (e.g., RNA, DNA) of the hybridizing regions of thenucleic acids can be considered in selecting hybridization conditions.An additional consideration is whether one of the nucleic acids isimmobilized, for example, on a filter.

An example of progressively higher stringency conditions is as follows:2×SSC/0.1% SDS at about room temperature (hybridization conditions);0.2×SSC/0.1% SDS at about room temperature (low stringency conditions);0.2×SSC/0.1% SDS at about 42° C. (moderate stringency conditions); and0.1×SSC at about 68° C. (high stringency conditions). Washing can becarried out using only one of these conditions, e.g., high stringencyconditions, or each of the conditions can be used, e.g., for 10-15minutes each, in the order listed above, repeating any or all of thesteps listed. However, as mentioned above, optimal conditions will vary,depending on the particular hybridization reaction involved, and can bedetermined empirically. In general, conditions of high stringency areused for the hybridization of the probe of interest.

Label

The probe of interest can be detectably labeled, for example, with aradioisotope, a fluorescent compound, a bioluminescent compound, achemiluminescent compound, a metal chelator, or an enzyme. Those ofordinary skill in the art will know of other suitable labels for bindingto the probe, or will be able to ascertain such, using routineexperimentation.

Kit

In accordance with the present invention, there is provided a kit usefulfor the detection of a cellular proliferative disorder in a subject.Kits according to the present invention include a carrier meanscompartmentalized to receive a sample therein, one or more containerscomprising a first container containing a reagent which sensitivelycleaves unmethylated cytosine, a second container containing primers foramplification of a CpG-containing nucleic acid, and a third containercontaining a means to detect the presence of cleaved or uncleavednucleic acid. Primers contemplated for use in accordance with theinvention include those set forth in SEQ ID NOS: 1-20, and anyfunctional combination and fragments thereof. Functional combination orfragment refers to its ability to be used as a primer to detect whethermethylation has occurred on the region of the genome sought to bedetected.

Carrier means are suited for containing one or more container means suchas vials, tubes, and the like, each of the container means comprisingone of the separate elements to be used in the method. In view of thedescription provided herein of invention methods, those of skill in theart can readily determine the apportionment of the necessary reagentsamong the container means. For example, one of the container means cancomprise a container containing methylation sensitive restrictionendonuclease. One or more container means can also be includedcomprising a primer complementary to the nucleic acid locus of interest.In addition, one or more container means can also be included containingan isoschizomer of the methylation sensitive restriction enzyme.

EXAMPLES

Hereinafter, the present invention will be described in further detailwith reference to examples. It is to be understood, however, that theseexamples are for illustrative purposes only and are not to be construedto limit the scope of the present invention.

Example 1 Discovery of Bladder Cancer-Specific Methylated Genes

In order to screen biomarkers which are methylated specifically inbladder cancer, about 20 ml of the urine of each of 10 bladder cancerpatients and 10 normal persons was centrifuged in a centrifuge (HanilScience Industrial Co., Ltd., Korea) at 4,200×g for 10 minutes toisolate urinary cells. The supernatant was discarded, and the cellprecipitate was washed twice with 5 ml of PBS. Genomic DNA was isolatedfrom the cell precipitate using the QIAamp DNA Mini kit (QIAGEN, USA).500 ng of the isolated genomic DNA was sonicated (Vibra Cell, SONICS),thus constructing about 200-300-bp-genomic DNA fragments.

To obtain only methylated DNA from the genomic DNA, a methyl bindingdomain (MBD) known to bind to methylated DNA (Fraga et al., Nucleic AcidRes., 31:1765-1774, 2003) was used. Specifically, 2 μg of 6×His-taggedMBD was pre-incubated with 500 ng of the genomic DNA of E. coli JM110(No. 2638, Biological Resource Center, Korea Research Institute ofBioscience & Biotechnology), and then bound to Ni-NTA magnetic beads(Qiagen, USA). 500 ng of the sonicated genomic DNA isolated from theurinary cells of the normal persons and the bladder cancer patients wasallowed to react with the beads in the presence of binding buffersolution (10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 3 mMMgCl₂, 0.1% Triton-X100, 5% glycerol, 25 mg/μl BSA) at 4° C. for 20minutes. Then, the beads were washed three times with 500 μl of abinding buffer solution containing 700 mM NaCl, and then methylated DNAbound to the MBD was isolated using the QiaQuick PCR purification kit(QIAGEN, USA).

Then, the methylated DNAs bound to the MBD were amplified using agenomic DNA amplification kit (Sigma, USA, Cat. No. WGA2), and 4 μg ofthe amplified DNAs were labeled with Cy3 for the normalperson-originated DNA and with Cy5 for the bladder cancerpatient-originated DNA using the BioPrime Total Genomic Labeling systemI (Invitrogen Corp., USA). The DNA of the normal persons and the DNA ofthe bladder patients were mixed with each other, and then hybridized to244K human CpG microarrays (Agilent, USA) (FIG. 1). After thehybridization, the DNA mixture was subjected to a series of washingprocesses, and then scanned using an Agilent scanner. The calculation ofsignal values from the microarray images was performed by calculatingthe relative difference in signal strength between the normal personsample and the bladder cancer patient sample using Feature Extractionprogram v. 9.5.3.1 (Agilent).

In order to select unmethylated spots from the normal sample, the wholeCy3 signal values were averaged, and then spots having a signal value ofless than 10% of the averaged value were regarded as those unmethylatedin the samples of the normal persons. As a result, 41,674 spots having aCy3 signal value of less than 65 were selected.

In order to select the methylated spots in the samples of the bladdercancer patients from among the 41,674 spots, spots having a Cy5 signalvalue of more than 130 were regarded as the methylated spots in bladdercancer. As a result, 631 spots having a Cy5 signal value of more than130 were selected. From these spots, 227 genes corresponding to thepromoter region were secured as bladder cancer-specific methylatedgenes.

From the genes, 10 genes (CDX2, CYP1B1, VSX16, HOXA11, T, TBX5, PENK,PAQR9, LHX2, and SIM2) showing the greatest relative difference betweenmethylation degree of the normal persons and that of the bladder cancerpatients were selected, and the presence of CpG islands in the promoterregion of the 10 genes was confirmed using MethPrimer(http://itsa.ucsfedu/˜urolab/methprimer/index 1.html). The 10 genes weresecured as methylation biomarkers for diagnosis of bladder cancer. Thelist of the 10 genes and the relative methylation degree thereof in theurinary cells of the bladder patients relative to those of the normalpersons are shown in Table 1 below.

TABLE 1 10 methylation biomarkers for diagnosis of bladder cancerBiomarker for bladder GenBank Relative cancer No. Descriptionmethylation ^(a) CDX2 NM_001265 caudal type homeobox 11.0 transcriptionfactor 2 CYP1B1 NM_000104 Cytochrome P450, family 1, 14.6 subfamily B,polypeptide 1 VSX1 NM_199425 visual system homeobox 1 33.4 homolog,CHX10-like (zebrafish) HOXA11 NM_005523 homeobox A11 14.2 T NM_003181 T,brachyury homolog 51.4 (mouse) TBX5 NM_080717 T-box 5 18.7 PENKNM_006211 Proenkephalin 12.7 PAQR9 NM_198504 progestin and adipoQ 4.1receptor family member IX LHX2 NM_004789 LIM Homeobox 2 5.8 SIM2 U80456Single-minded homolog 2 9.5 (Drosophila) ^(a) Relative methylationdegree between the normal sample and the bladder patient sample,calculated by dividing the average signal (Cy5) value in the bladdercancer patient sample in CpG microarrays by the average signal (Cy5)value in the normal person sample.

Example 2 Measurement of Methylation of Biomarker Genes in Cancer CellLines

In order to further determine the methylation status of the 10 genes,bisulfite sequencing for each promoter was performed.

In order to modify unmethylated cytosine to uracil using bisulfite,total genomic DNA was isolated from the bladder cancer cell lines RT-4(Korean Cell Line Bank (KCLB 30002), J82 (KCLB 30001), HT1197 (KCLB21473) and HT1376 (KCLB 21472), and 200 ng of the genomic DNA wastreated with bisulfite using the EZ DNA methylation-gold kit (ZymoResearch, USA). When DNA is treated with bisulfite, unmethylatedcytosine is modified to uracil, and the methylated cytosine remainswithout changes. The DNA treated with bisulfite was eluted in 20 μl ofsterile distilled water and subjected to pyrosequencing.

PCR and sequencing primers for performing pyrosequencing for the 10genes were designed using the PSQ assay design program (Biotage, USA).The PCR and sequencing primers for measuring the methylation of eachgene are shown in Tables 2 and 3 below.

TABLE 2 Primers and conditions SEQ ID CpG Amplicon Gene PrimerSequence (5′→3′) NO: position^(a) size CDX2 forwardTGGTGTTTGTGTTATTATTAATAG 1 −138, −129, 129 bp reverseBiotin-CACCTCCTTCCCACTAAACTA 2 −121, −118 CYP1B1 forwardGTAAGGGTATGGGAATTGA 3 +73, +83,  90 bp reverseBiotin-CCCTTAAAAACCTAACAAAATC 4 +105 VSX1 forward GGAGTGGGATTGAGGAGATTT5 −1121, −1114,  89 bp reverse Biotin-AAACCCAACCAACCCTCAT 6 −1104, 1100HOXA11 forward AGTAAGTTTATGGGAGGGGGATT 7 −415, −405 243 bp reverseBiotin- 8 −388 CCCCCATACAACATACTTATACTCA T forwardGGAGGAATGTTATTGTTTAAAGAGAT 9 −95, −89, 326 bp reverseBiotin-CAACCCCTTCTAAAAAATATCC 10 −76, −71, −69 TBX5 forwardGGGTTTGGAGTTAGGTTATG 11 −645, −643,  95 bp reverseBiotin-AAATCTAAACTTACCCCCAACT 12 −628, −621 PENK forwardATATTTTATTGTATGGGTTTTTTAATAG 13 −150 −148, 322 bp reverseBiotin-ACAACCTCAACAAAAAATC 14 −139, −135,  54 bp −133 PAQR9 forwardBiotin-AGATAGGGGATAATTTTAT 15 −480, −475,  54 bp reverseCCTCCCAAACTAAAATTT 16 −471, −469 LHX2 forward GTAGAAGGGAAATAAGGTTGAAA 17+5093, 233 bp reverse Biotin-ACTAAAACCCCAATACTCCCA 18 +5102, +5113,+5125, +5127 SIM2 forward Biotin-GTGGATTTAGATTAGGATTTTGT 19−6776, −6774, 205 bp reverse CACCCTCCCCAAATTCTT 20 −6747, −6744, −6743^(a)distances (nucleotides) from the transcription initiation site (+1):the positions of CpG regions on the genomic DNA used in the measurementof methylation

TABLE 3 Sequences of sequencing primers for methylation marker genesGene Sequence (5′ --> 3′) SEQ ID NO: CDX2 ATT AAT AGA GTT TTG TAA ATA T21 CYP1B1 AAG GGT ATG GGA ATT G 22 VSX1 TTT GGG ATT GGG AAG 23 HOXA11TAG TTT AGG GTA TTT TTT ATT TAT 24 T GTG AAA GTA ATG ATA TAG TAG AAA 25TBX5 TTT GGG GGT TGG GGA 26 PENK GGG TGT TTTAGG TAG TT 27 PAQR9CCT CCC AAA CTA AAA TTT C 28 LHX2 TGG GGG TAG AGG AGA 29 SIM2CCT CCC CAA ATT CTT C 30

20 ng of the genomic DNA modified with bisulfite was amplified by PCR.In the PCR amplification, a PCR reaction solution (20 ng of the genomicDNA modified with bisulfite, 5 μl of 10×PCR buffer (Enzynomics, Korea),5 units of Taq polymerase (Enzynomics, Korea), 4 μl of 2.5 mM dNTP(Solgent, Korea), and 2 μl (10 pmole/μl) of PCR primers) was used, andthe PCR reaction was performed in the following conditions:predenaturation at 95° C. for 5 min, and then 45 cycles of denaturationat 95° C. for 40 sec, annealing at 60° C. for 45 sec and extension at72° C. for 40 sec, followed by final extension at 72 r for 5 min. Theamplification of the PCR product was confirmed by electrophoresis on2.0% agarose gel.

The amplified PCR product was treated with PyroGold reagents (Biotage,USA), and then subjected to pyrosequencing using the PSQ96MA system(Biotage, USA). After the pyrosequencing, the methylation degree of theDNA was measured by calculating the methylation index. The methylationindex was calculated by determining the average rate of cytosine bindingto each CpG island.

FIG. 2 quantitatively shows the methylation degree of the 10 biomarkersin the bladder cancer cell lines, measured using the pyrosequencingmethod. As a result, it was shown that the 10 biomarkers were allmethylated at high levels in at least one of the cell lines. Table 4below shows the promoter sequences of the 10 genes.

TABLE 4 Promoter sequences of methylation marker genes Gene SEQ ID NO:CDX2 31 CYP1B1 32 VSX1 33 HOXA11 34 T 35 TBX5 36 PENK 37 PAQR9 38 LHX239 SIM2 40

Example 3 Measurement of Methylation of Biomarker Genes in Urinary Cellsof Bladder Cancer Patients

In order to verify whether the 10 genes can be used as biomarkers fordiagnosis of bladder cancer, about 20 ml of the urine of each of 20normal persons and 19 bladder cancer patients was centrifuged in acentrifuge (Hanil Science Industrial Co., Ltd., Korea) at 4,200×g for 10minutes to isolate cells. The supernatant was discarded, and the cellprecipitate was washed twice with 5 ml of PBS. Genomic DNA was isolatedfrom the washed cells using the QIAamp DNA Mini kit (QIAGEN, USA), and200 ng of the isolated genomic DNA was treated with bisulfite using theEZ DNA methylation-Gold kit (Zymo Research, USA). Then, the DNA waseluted in 20 μl of sterile distilled water and subjected topyrosequencing.

20 ng of the genomic DNA converted with bisulfite was amplified by PCR.In the PCR amplification, a PCR reaction solution (20 ng of the genomicDNA modified with bisulfite, 5 μl of 10×PCR buffer (Enzynomics, Korea),5 units of Taq polymerase (Enzynomics, Korea), 4 μl of 2.5 mM dNTP(Solgent, Korea), and 2 μl (10 pmole/μl) of PCR primers) was used, andthe PCR reaction was performed in the following conditions:predenaturation at 95° C. for 5 min, and then 45 cycles of denaturationat 95° C. for 40 sec, annealing at 60° C. for 45 sec and extension at72° C. for 40 sec, followed by final extension at 72 r for 5 min. Theamplification of the PCR product was confirmed by electrophoresis on2.0% agarose gel.

The amplified PCR product was treated with PyroGold reagents (Biotage,USA), and then subjected to pyrosequencing using the PSQ96MA system(Biotage, USA). After the pyrosequencing, the methylation degree of theDNA was measured by calculating the methylation index thereof. Themethylation index was calculated by determining the average rate ofcytosine binding to each CpG region. After the methylation index of DNAin the urinary cells of the normal persons and the bladder cancerpatients has been measured, a methylation index cut-off value fordiagnosis of bladder cancer patients was determined through receiveroperating characteristic (ROC) curve analysis.

FIGS. 3(A)-3(D) show measurement results for the methylation of the 10biomarker genes in urinary cells. As can be seen in FIG. 3, themethylation degree of the genes was higher in the sample of the bladdercancer patients than in the sample of the normal persons. Meanwhile, themethylation index in the cystitis patients and the hematuria patientswas similar to that in the normal control group or was rarely higherthan that in the normal control group. FIGS. 4( a)-4(e) show ROCanalysis results for determining cut-off values for diagnosis of bladdercancer. Also, methylation index cut-off values for the 10 biomarkers,calculated based on the ROC curve analysis results, are shown in Table 5below.

TABLE 5 Cut-off values for bladder cancer diagnosis of 10 biomarkersGene cut-off (%) ^(a) CDX2 5.82< CYP1B1 8.38< VSX1 29.3< HOXA11 8.81< T11.3< TBX5 6.93< PENK 11.57< PAQR9 5.0< LHX2 13.7< SIM2 8.2<

In the analysis of the methylation of the 10 biomarkers, the methylationindex of each biomarker in the clinical sample was calculated. The casein which the calculated methylation index for diagnosis of bladdercancer was higher than the cut-off value obtained through receiveroperating characteristic (ROC) analysis was judged to bemethylation-positive, and the case in which the calculated methylationindex was lower than the cut-off value was judged to bemethylation-negative.

As shown in Table 6 below and FIG. 5, when judged on the basis of thecut-off value obtained by ROC curve analysis, the urinary cells of thenormal persons were methylation-negative for all the 10 biomarkers, but12.5-62.5% of the samples of the bladder cancer patients weremethylation-positive for the 10 biomarkers. Also, statistical analysiswas performed and, as a result, it could be seen that 9 of the samplesof the bladder cancer samples were methylation-positive for 9 of the 10biomarkers at a significant level (p<0.01) compared to the normal persongroup. This suggests that 9 of the 10 methylation markers arestatistically significantly methylated specifically in bladder cancerand are highly useful for diagnosing bladder cancer.

TABLE 6 Frequency of methylation-positive samples for 10 biomarkers No.of methylation- positive samples/No. of total samples (%) ^(a) GeneNormal bladder cancer patient P value^(b) CDX2 0/31 (0) 9/32 (28.1)0.002 CYP1B1 0/31 (0) 16/32 (50.0) <0.001 VSX1 0/31 (0) 14/32 (45.2)<0.001 HOXA11 0/31 (0) 17/32 (53.1) <0.001 T 0/31 (0) 15/32 (46.9)<0.001 TBX5 0/31 (0) 20/32 (62.5) <0.001 PENK 0/31 (0) 19/32 (59.4)<0.001 PAQR9 0/31 (0) 4/32 (12.5) 0.113 LHX2 0/17 (0) 13/24 (54.2)<0.001 SIM2 0/17 (0) 15/24 (62.5)0 <0.001 ^(a)frequency ofmethylation-positive samples; and ^(b)p values obtained through theChi-Square test

Example 4 Evaluation of the Ability of 6 Biomarker Panel Genes toDiagnose Bladder Cancer

Using the 10 methylation biomarkers, logistic regression analysis wasperformed. As a result, an optimal panel of 6 genes for diagnosingbladder cancer was established. FIG. 6A shows the methylation status ofthe 6 biomarkers (CYP1B1, HOXA11, SIM2, PENK, LHX2 and TBX5). Whethersamples were methylation-positive or methylation-negative for the 6genes was judged according to the method described in Example 3. As aresult, it could be seen that all the normal samples weremethylation-negative for the 6 genes, and only the bladder cancersamples were methylation-positive for the 6 genes. Particularly, earlybladder cancer samples were also methylation-positive for the 6 genes ata high frequency, suggesting that the 6 genes are highly useful forearly diagnosis of bladder cancer. When the methylation of at least onegene of the gene panel consisting of the six genes was diagnosed asbladder cancer, the sensitivity and specificity of the gene panel forearly bladder cancer were as extremely high as 84.0% and 100%,respectively (FIG. 6D). Also, the sensitivity and specificity of thegene panel for advanced bladder cancer were measured to be 85.7% and100%, respectively (FIG. 6C). In addition, the sensitivity andspecificity of the gene panel for all early and advanced bladder cancerswere measured to be 84.4% and 100%, respectively (FIG. 6B). Thissuggests that the methylation of the 6 genes is highly useful for earlydiagnosis of bladder cancer.

Example 5 Measurement of Methylation of Biomarker Genes Using MethylatedDNA-Specific Binding Protein

In order to measure the methylation of biomarkers which are methylatedspecifically in bladder cancer, 100 ng of the genomic DNA of each of thebladder cancer cell lines RT24 and HT1197 was sonicated (Vibra Cell,SONICS), thus obtaining about 200-400-bp genomic DNA fragments.

To obtain only methylated DNA from the genomic DNA, MBD known to bind tomethylated DNA was used. Specifically, 2 μg of 6×His-tagged MBD waspre-incubated with 500 ng of the genomic DNA of E. coli JM110 (No. 2638,Biological Resource Center, Korea Research Institute of Bioscience &Biotechnology), and then bound to Ni-NTA magnetic beads (Qiagen, USA).100 ng of the sonicated genomic DNA was allowed to react with the beadsin the presence of binding buffer solution (10 mM Tris-HCl (pH 7.5), 50mM NaCl, 1 mM EDTA, 1 mM DTT, 3 mM MgCl₂, 0.1% Triton-X100, 5% glycerol,25 mg/μl BSA) at 4° C. for 20 minutes. Then, the beads were washed threetimes with 500 μl of a binding buffer solution containing 700 mM NaCl,and then methylated DNA bound to the MBD was isolated using the QiaQuickPCR purification kit (QIAGEN, USA).

Then, the DNA methylated DNA bound to the MBD was amplified by PCR usingprimers of SEQ ID NOS: 41 and 42 corresponding to the promoter region(from −6842 to −6775 bp) of the SIM2 gene.

SEQ ID NO: 41: 5′-TTC TTA TTC TCA CCA GAC ATC TCA ACA CCC-3′SEQ ID NO: 42: 5′-ATC TCC CAT CCT CCC TCC CAC TCT C-3′

The PCR reaction was performed in the following condition:predenaturation at 94° C. for 5 min, and then 40 cycles of denaturationat 94° C. for 30 sec, annealing at 62° C. for 30 sec and extension at72° C. for 30 sec, followed by final extension at 72° C. for 5 min. Theamplification of the PCR product was confirmed by electrophoresis on 2%agarose gel.

As a result, it was seen that, for the SIM2 gene, a 168-bp amplifiedproduct was detected only in the genomic DNA of the RT24 cell line,suggesting that the gene was methylated, whereas no amplified productwas detected in the HT1197 cell line, suggesting that the gene was notmethylated in the HT1197 cell line (FIG. 7). Such results wereconsistent with the methylation measurement results obtained by thepyrosequencing method. Also, such results indicate that the use of MBDenables detection of methylated DNA.

INDUSTRIAL APPLICABILITY

As described above in detail, the present invention provides a kit andnucleic acid chip for diagnosing bladder cancer, which can detect themethylation of CpG islands of bladder cancer-specific marker genes. Itis possible to diagnose bladder cancer at an early stage oftransformation using the diagnostic kit or nucleic acid chip of thepresent invention, thus enabling early diagnosis of bladder cancer, andthe diagnostic kit or nucleic acid chip can diagnose bladder cancer in amore accurate and rapid manner compared to a conventional method.

Although the present invention has been described in detail withreference to the specific features, it will be apparent to those skilledin the art that this description is only for a preferred embodiment anddoes not limit the scope of the present invention. Thus, the substantialscope of the present invention will be defined by the appended claimsand equivalents thereof.

1.-8. (canceled)
 9. A method for detecting bladder carcinoma or bladdercell proliferative disorder, the method comprising the step of: (a)examining a CpG methylation of a clinical sample-originatedPENK—proenkephalin gene; and (b) detecting bladder carcinoma or bladdercell proliferative disorder based on increased CpG methylation of thePENK gene, relative to that of a control.
 10. The method for detectingbladder carcinoma or bladder cell proliferative disorder according toclaim 9, wherein the step (a) is selected from the group consisting ofPCR, methylation specific PCR, real-time methylation specific PCR, PCRusing a methylated DNA-specific binding protein, quantitative PCR,pyrosequencing, and bisulfite sequencing
 11. The method for detectingbladder carcinoma or bladder cell proliferative disorder according toclaim 9, wherein the clinical sample is tissue, cell, blood, urine,serum or plasma.
 12. The method for detecting bladder carcinoma orbladder cell proliferative disorder according to claim 9, wherein step(a) comprises examining a CpG methylation of a promoter or exon regionof the clinical sample-originated PENK.
 13. The method for detectingbladder carcinoma or bladder cell proliferative disorder according toclaim 12, wherein the promoter is a DNA sequence represented in SEQ IDNO:
 37. 14. The method for detecting bladder carcinoma or bladder cellproliferative disorder according to claim 9, wherein the method furthercomprises the step of examining CpG methylation of a clinicalsample-originated gene selected from the group consisting of TBX5—T-box5; CDX2—caudal type homeobox transcription factor 2; CYP1B1—cytochromeP450, family 1, subfamily B, polypeptide 1; VSX1—visual system homeobox1 homolog, CHX10-like (zebrafish); HOXA11—homeobox A11; T—T, brachyuryhomolog (mouse); PAQR9—progestin and adipoQ receptor family member IV;and LHX2—LIM Homeobox
 2. 15. The method for detecting bladder carcinomaor bladder cell proliferative disorder according to claim 14, whereinthe step of examining comprises examining CpG methylation of a promoteror exon region of the clinical sample-originated gene selected from thegroup consisting of TBX5; CDX2; CYP1B1; VSX1; HOXA11; T; PAQR9; andLHX2.
 16. The method for detecting bladder carcinoma or bladder cellproliferative disorder according to claim 9, wherein the method furthercomprises the step of contacting at least one nucleic acid isolated fromthe clinical sample with an agent capable of determining a CpGmethylation status of PENK gene.
 17. The method for detecting bladdercarcinoma or bladder cell proliferative disorder according to claim 9,wherein step (a) comprises: contacting a nucleic acid-containingclinical sample isolated from the clinical sample with an agent thatdistinguishes between methylated and non-methylated CpG dinucleotidesfor determination of a CpG methylation status; contacting the treatednucleic acid with an amplification enzyme and a primer set; andidentifying the methylation state of a CpG methylation of the clinicalsample-originated PENK gene by detecting whether the treated nucleicacid is either amplified to produce at least one amplificate, or is notamplified.
 18. The method for detecting bladder carcinoma or bladdercell proliferative disorder according to claim 17, wherein the agentthat distinguishes between methylated and non-methylated CpGdinucleotides is a reagent selected from the group consisting ofbisulfite, methylation sensitive restriction enzyme, and methylatedDNA-specific binding peptide.
 19. The method for detecting bladdercarcinoma or bladder cell proliferative disorder according to claim 9,wherein step (a) comprises: digesting a nucleic acid isolated from theclinical sample with a methylation sensitive restriction enzyme;contacting the digested nucleic acid with an amplification enzyme and atleast two primers suitable for the amplification of a sequencecomprising at least one CpG dinucleotide of the clinicalsample-originated PENK gene; and identifying the methylation state of aCpG methylation of the clinical sample-originated PENK gene based on apresence or absence of an amplificate.
 20. The method for detectingbladder carcinoma or bladder cell proliferative disorder according toclaim 9, wherein step (a) comprises using a kit comprising primerscapable of detecting a methylated region of the bladder cancer markergene PENK; and a methylation detection reagent selected from the groupconsisting of bisulfite, methylation sensitive restriction enzyme andmethylated DNA-specific binding protein.
 21. The method for detectingbladder carcinoma or bladder cell proliferative disorder according toclaim 9, wherein step (a) comprises using a nucleic acid chip comprisinga probe capable of hybridizing with a fragment containing a CpG islandof the bladder cancer marker gene PENK.
 22. The method for detectingbladder carcinoma or bladder cell proliferative disorder according toclaim 9, wherein the clinical sample is originated from patientssuspected of cancer or subjects to be diagnosed.